Diphtheria toxin (DT) has been deeply studied. The studies on structure show that diphtheria toxin consists of three domains: N-terminal Catalytic Domain C (aa 1-190, C domain) (also called Fragment A), middle Transmembrane Domain T (aa 201-384, T domain), and C-terminal Receptor Binding domain R (aa 386-535, R domain) (Choe S, Bennett M, Fujii G, et al., Nature. 1992. 357:216-222). ONTAK (DAB389-IL-2), prepared by fusion of the former two domains of diphtheria toxin with interleukin 2 (IL-2), was approved by FDA on the market in 1999, for the treatment of adult cutaneous T-cell lymphoma. This demonstrates that the three domains of diphtheria toxin may be used separately and play their own roles, respectively.
CRM197 (Cross-Reacting Materials 197) is a diphtheria toxin non-toxic mutant (Uchida, T., A. M, Pappenheimer, Jr., R. Gregory, et al., J. Biol. Chem. 1973. 248:3838-3844), which differs from a wild-type gene encoding DT by a single nucleotide mutation, resulting in the amino acid residue at position 52 changed from Gly to Glu (G. Giannini, R. Rappuoli, G. Ratti et al., Nucleic Acids Research. 1984. 12: 4063-4070).
Studies show that although CRM197 has a structure similar to that of a wild-type DT (namely, having said three domains), its Fragment A loses the ability of binding to NAD, is unable to bind to EF2 and thereby loses the cytoxicity possessed by natural DT, indicating that the amino acid residue Gly at position 52 plays an important role in the binding of DT to NAD (K. Moyner, G. Christiansen, Acta path microbial immunolscand sect C. 1984, 92:17-23). Although CRM197 loses the cytotoxicity, it retains a strong immunogenicity comparable to that of a wild-type DT. Therefore, CRM197 is generally used as a protein carrier for crosslinking other haptens so as to prepare conjugate vaccines.
As early as 1985, Porter et al., crosslinked polysaccharides on Hib surface to CRM197 and DT protein carrier, respectively, and prepared them into vaccines, and studied the difference of them in immunogenicity. The experimental results showed that there was no significant difference between the two crosslinked vaccines in terms of immune effect, both of them could stimulate the generation of a strong immune response and immunologic memory in infants (Porter Anderson, Micheal E. Pichichero and Richard A. J. Clin. Invest. 1985: 52-59). After comparing pneumococcal conjugate vaccines crosslinked to various proteins, it is found that the vaccines wherein CRM197 is used as a protein carrier have a good immune effect in animal experiments and clinical trials, and CRM197 is safe without a side-effect of toxicity (Black, S., H. Shinefield, et al. Pediatr Infect Dis J, 2000, 19(3); 187-195). In current, pneumococcal conjugate vaccines, in which CRM197 is used as a protein carrier, mainly refer to PCV7, PCV9, PCV13, and the like. The results of clinical trials showed that these vaccines had good immunogenicity and safety in children less than two years old (Barricarte, A., J. Castilla, et al. Clin Infect Dis, 2007, 44(11): 1436-1441; Madhi, S., P. Adrian, et al. Vaccine, 2007, 25(13): 2451-2457; Duggan, S. T. Drugs, 2010, 70(15): 1973-1986). Epidemic meningitis conjugate vaccines can be prepared by crosslinking CRM197 to polysaccharides on surface of N. menigitidis. For example, vaccines such as Meningitec (Wyeth Pharmaceuticals), Menjugate (Novartis vaccines), and Menveo (Novartis vaccines), in which CRM197 is used as a protein carrier, have been commercially available.
Although CRM197 loses enzymatic activity and cytotoxity, it is still able to bind to a specific receptor of DT, i.e. heparin-binding EGF-like growth factor (HB-EGF). Since the expression of the receptor is generally up-regulated in cancerous tissues, like DT, CRM197 also has anti-tumor effect (Buzzi, S., D. Rubboli, et al. Immunotherapy, 2004, 53(11)). The studies also found that CRM197 could pass through Blood-Brain-Barrier (BBB), and therefore could be used as a carrier for delivery of drugs to brain (Gaillard, P. J., and A. G. de Boer. J Control Release, 2006, 116(2): 60-62).
Although it has been reported that CRM197 has multiple functions, in particular, has a strong immunogenicity and can be used as immunoadjuvant, it is not reported yet that CRM197 may be used as intramolecular adjuvant for enhancing immunogenicity of a target protein fused thereto in a fusion protein. The invention uses Hepatitis E capsid protein as an example, and demonstrates for the first time that CRM197 or a fragment thereof can enhance immunogenicity of a protein fused thereto in a fusion protein, and thereby can be used as intramolecular adjuvant.